1. Field of the Invention
The present invention relates to an improved method for multiplex PCR for resulting in at least two amplified DNA products from one PCR solution. In particular, the present invention relates to the novel method for performing multiplex PCR to solve the problems that size differences among many types of required amplified products can not be greatly increased, non-specific DNA amplified products are produced, and dimers caused by using at least two pairs of primers in the conventional multiplex PCR.
2. Description of the Prior Art
To perform disease diagnosis, detection and identification of pathogenic bacteria, and research on variation or abnormality of organism gene, such as SNP(single nucleotide polymorphism), STR(short tandem repeat), insertion, deletion and mutation etc, the sequence of amplified target DNA must be secured in advance. As a method for amplifying target DNA sequence, there exists PCR (polymerase chain reaction) method, SDA(strand displacement amplification) method, and ones for TMA(transcription mediated amplification), by which the target DNA is synthesized from mRNA, NASBA(nucleic acid sequence based amplification) and so on. In general applications, the PCR method is widely used for its amplification efficiency superior to other methods above.
In recent years, researches have been conducted to perform DNA amplification in a small equipment or a small chip like a lab-on-a-chip and to analyze DNAs in a short tome
However, common problems of the above methods are have common problems as follows. It is complicated to purely refine genomes from blood samples or tissue samples, and various target genes must be amplified safely in a small space like chips in order to obtain information as many as possible with small costs. Multiplex PCR method have been used to obtain various DNA amplified products in a limited space.
The multiplex PCR method produces at least two amplified DNA products from one reaction solution, and at least two pairs of primers are used. Therefore, if the reaction condition and primer sequence are not appropriate, primer dimers may be produced or non-specific DNA products may be produced, or required-amplified DNA products can not be obtained. Conventional methods require a lot of times and efforts in order to find the optimal condition for amplification of different size of DNA because temperature and time conditions are fixed. For example, in the documents by Hinegariu O et al, “Multiplex PCR: critical parameters and step-by-step protocol”(Biotechniques, 1997, pp. 504–511) and by P. markoulatos et al, “Multiplex Polymerase Chain Reaction: a practical approach” (J. Clin. Lab. Anal. 2002. pp. 47–51), researches have been conducted to find the optimal multiplex PCR conditions for various combination of PCR parameters like concentration of primers, Mg++, taq DNA polymerase buffer, temperature of annealing and extension time to a specific sample. However, these conventional methods have some problems, such as, the methods require long time, much efforts and large amount of samples to optimize the PCR condition because of various combinations of PCR parameter, and the procedures should be repeated for a new samples.